The telomerase ribonucleoprotein reverse transcriptase adds telomeric repeats to chromosome ends. This unusual mode of DMAsynthesis counteracts the terminal sequence loss that occurs upon genome replication by DNA-templated DNA polymerases. Most eukaryotic cells have few chromosomes, few telomeres and little telomerase. Ciliated protozoa are the exception: they possess thousands to millions of telomeres per cell and high levels of telomerase. This proposal capitalizes on advantages of the ciliate Tetrahymena thermophila for studies of telomerase. Telomerase abundance facilitates direct biochemical assays of endogenously assembled enzyme. Studies of Tetrahymena telomerase are also aided by unparalleled in vitro reconstitution assays and robust methods for molecular genetic manipulation in vivo. Endogenously assembled Tetrahymena and human telomerases show remarkably similar biochemical properties, particularly when compared to telomerases from other model organisms. Thus, studies of Tetrahymena telomerase will continue to inform studies of the less experimentally tractable human enzyme. Our understanding of telomerase is limited by scant knowledge of enzyme structure and by the difficulty of identifying and characterizing factors required for endogenous enzyme function. Filling these knowledge gaps is the long-term goal of this proposal. Following from our previous in vitro reconstitution studies, Aim I describes recombinant RNA and protein structural studies using site-specific cross-linking, fluorescence resonance energy transfer and crystallography. Following from our previous affinity purification of an endogenously assembled telomerase holoenzyme, Aims II, III and IV use in vitro and in vivo reconstitution assays to characterize the biochemical and biological activities of four new holoenzyme proteins. Insights about telomerase structure and function have direct relevance for the improvement of human health. Most human somatic cells lack functional telomerase and therefore have a capacity for division that is limited by telomere erosion. In diseases characterized by premature telomere loss, temporary telomerase activation could restore cellular renewal potential. Cancer cells aberrantly activate telomerase to gain extended cell division capacity and require telomerase for their continued viability. For this reason, telomerase inhibitors could be broadly effective cancer therapeutics.